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Nat Rev Microbiol. Author manuscript; available in PMC 2010 July 27.
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Nat Rev Microbiol. 2009 February ; 7(2): 99109. doi:10.1038/nrmicro2070.
Pyroptosis: host cell death and inflammation
Tessa Bergsbaken*, Susan L. Fink, and Brad T. Cookson*,

*Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.
Department of Molecular and Cellular Biology, University of Washington, Seattle, Washington

98195, USA.
Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195, USA.
Abstract
Eukaryotic cells can initiate several distinct programmes of self-destruction, and the nature of the
cell death process (non-inflammatory or proinflammatory) instructs responses of neighbouring
cells, which in turn dictates important systemic physiological outcomes. Pyroptosis, or caspase 1-
dependent cell death, is inherently inflammatory, is triggered by various pathological stimuli, such
as stroke, heart attack or cancer, and is crucial for controlling microbial infections. Pathogens have
evolved mechanisms to inhibit pyroptosis, enhancing their ability to persist and cause disease.
Ultimately, there is a competition between host and pathogen to regulate pyroptosis, and the
outcome dictates life or death of the host.
Cells can die through distinct biochemical pathways that produce different morphological
and physiological outcomes. Apoptosis is perhaps the most widely recognized programme
of cell death, and is mechanistically defined by the requirement for particular cysteine-
dependent aspartate-specific proteases, or caspases, which produce an orchestrated
disassembly of the cell1. Apoptotic caspases cleave cellular substrates, resulting in the
characteristic features of apoptosis, which include cytoplasmic and nuclear condensation,
DNA cleavage and maintenance of an intact plasma membrane. The contents of apoptotic
cells are packaged into membrane-enclosed apoptotic bodies, which are targeted for
phagocytosis and removal in vivo, resulting in an absence of inflammation2 (BOX 1).
Although apoptosis was the first well-recognized programme of eukaryotic cell death,
programmed cell death is broadly applied to several endogenous genetically defined
pathways in which the cell plays an active part in its own destruction3. Other cell death
programmes include autophagy, oncosis and caspase 1-dependent programmed cell death
(also known as pyroptosis). Pyroptosis is a more recently identified pathway of host cell
death that is stimulated by a range of microbial infections (for example, Salmonella,
Francisella and Legionella) and non-infectious stimuli, including host factors produced
during myocardial infarction4. Caspase 1 was first recognized as a protease that processes
the inactive precursors of interleukin 1 (IL-1) and IL-18 into mature inflammatory
cytokines, and was initially called interleukin IL-1-converting enzyme5. However, caspase
1 activation can result not only in the production of activated inflammatory cytokines, but
also rapid cell death characterized by plasma-membrane rupture and release of
proinflammatory intracellular contents6,7. Caspase 1-dependent cell death is a programmed
process of cellular self-destruction mediated by caspases, and therefore was not initially
distinguished from apoptosis811. However, the mechanism, characteristics and outcome of
caspase 1-dependent cell death are distinct from apoptosis6,7,12. Thus, the term pyroptosis
Correspondence to B.T.C. cookson@u.washington.edu.

Bergsbaken et al. Page 2
(from the Greek pyro, relating to fire or fever, and ptosis, meaning a falling (BOX 1)), is
used to described the inherently inflammatory process of caspase 1-dependent programmed
cell death13.
Mechanism and features of pyroptosis

Pyroptosis is morphologically and mechanistically distinct from other forms of cell death.
Caspase 1 dependence is a defining feature of pyroptosis, and caspase 1 is the enzyme that
mediates this process of cell death (FIG. 1). Caspase 1 is not involved in apoptosis, and
caspase 1-deficient mice have no defects in apoptosis and develop normally14,15. The
apoptotic caspases, including caspase 3, caspase 6 and caspase 8, are not involved in
pyroptosis6,10,12,1620, and substrates of apoptotic caspases, including poly (ADP-ribose)
polymerase and inhibitor of caspase-activated DNase (ICAD), do not undergo proteolysis
during pyroptosis6,7,9,12. Furthermore, loss of mitochondrial integrity and release of
cytochrome c, which can activate apoptotic caspases, do not occur during pyroptosis16,19.
Pyroptosis features rapid plasma-membrane rupture and release of proinflammatory

intracellular contents. This is in marked contrast to the packaging of cellular contents and
non-inflammatory phagocytic uptake of membrane-bound apoptotic bodies that
characterizes apoptosis2. Cell lysis during pyroptosis results from caspase 1-mediated
processes8,9,12,17,18,2024. Salmonella infection or treatment with lethal toxins from
Bacillus anthracis produces plasma-membrane pores with a functional diameter of 1.12.4

nm7,20, and pore formation is a host cell-mediated process that is dependent on caspase 1
activity7,12,20. Caspase 1-dependent plasma-membrane pores dissipate cellular ionic
gradients, producing a net increased osmotic pressure, water influx, cell swelling and,
eventually, osmotic lysis and release of inflammatory intracellular contents7. Indeed, cells
dying by pyroptosis undergo a measurable size increase7,18 (FIG. 1). In support of this
mechanism, the cytoprotective agent glycine non-specifically blocks ion fluxes in damaged
eukaryotic cells and thereby prevents swelling and lysis during pyroptosis6,7,21,25,26.
Cleavage of chromosomal DNA is a fatal event that is often assumed to indicate apoptotic
cell death3; however, DNA damage also occurs during pyroptosis6,12,24,27,28. During
apoptosis, caspase-mediated proteolysis of ICAD releases caspase-activated DNase (CAD).
CAD cleaves DNA between nucleosomes, resulting in characteristic oligonucleosomal DNA
fragments of approximately 180 bp7. Although purified caspase 1 can cleave ICAD in
vitro11, ICAD degradation does not occur during pyroptosis7,12. DNA cleavage during
pyroptosis instead results from the activity of an unidentified caspase 1-activated nuclease
that does not produce the oligonucleosomal DNA fragmentation pattern that is characteristic
of apoptosis7,12,29. DNA cleavage is accompanied by marked nuclear condensation, but
unlike apoptosis, nuclear integrity is maintained12,23 (FIG. 1). DNA cleavage and cell lysis
are both caspase 1-dependent features of pyroptosis, but cell lysis does not require DNA
cleavage7.
Destruction of the actin cytoskeleton has also been observed in cells undergoing pyroptosis,
but the mechanism and importance of this destruction remains unclear12,26. Caspase 1-
dependent degradation of cellular inhibitor of apoptosis protein (cIAP) also accompanies
during pyroptosis, although the exact mechanism that underlies cIAP degradation is also
unknown30. Caspase 1 cleaves and inactivates metabolic enzymes during pyroptosis31, and
identification of additional proteolytic targets of caspase 1 could yield insight into the
mechanism of pyroptosis and novel features of this form of cell death.
Box 1 | Apoptosis is a programmed process that results in non-inflammatory cell

death
Hippocrates was the first to use the term apoptosis in the medical literature
(approximately 460370 BCE)121. After years of exhaustive microscopic evaluation,
apoptosis was reintroduced by Kerr et al. in 1972 to describe an active, programmed
process that leads to cell death in both healthy and diseased tissues122. Its morphological
characteristics included condensation of both the cytoplasm and the nucleus, and the
generation of cell fragments called apoptotic bodies, which were phagocytosed by intact
cells and subsequently destroyed. Little tissue disruption and a marked lack of
inflammation suggested the process was a general mechanism of controlled cell
deletion, which is complementary to mitosis in the regulation of animal cell populations.
(REF. 122) Cell death caused by apoptosis was previously referred to as shrinkage
necrosis. By contrast, coagulative necrosis was invariably caused by noxious stimuli
and resulted from irreversible disturbance of cellular homeostatic mechanisms. (REF.
122) These original descriptions are consistent with recent recommendations for using
nomenclature that defines cell death, or necrosis, as the end product of processes such as
apoptosis3,123. The term apoptosis, which in Greek is used to describe the falling off
of leaves from a tree, was suggested to indicate the controlled loss of individual cells
from the population. Pronunciation provides a clear indication of its Greek roots: we
propose that the stress should be on the penultimate syllable, the second half of the word
being pronounced like ptosis (with the p silent), which comes from the same root to
fall and is already used to describe drooping of the upper eyelid. (REF. 122) The
ultrastructural features described in this landmark paper are still considered to be
hallmarks of apoptosis, and subsequent research has identified the important role of a
subset of caspases in mediating the morphological changes observed in this and other
early studies1.
TLRs and NLRs

The host can use a range of mechanisms to sense intracellular and extracellular danger
signals generated by invading pathogenic microorganisms or by the host in response to
tissue injury32. Toll-like receptors (TLRs) initiate a signalling cascade that leads to cellular
activation and production of inflammatory cytokines, such as tumour necrosis factor (TNF),
IL-6, IL-8 and type I interferons (IFNs), in response to extracellular signals33 (FIG. 2). Nod-
like receptors (NLRs) function in the recognition of danger signals introduced into the host
cell cytosol34. The NLRs nucleotide-binding oligomerization domain-containing protein 1
(NOD1) and NOD2 trigger a signalling cascade following ligand recognition that, similarly
to the cascade initiated by TLRs, results in inflammatory cytokine production34 (FIG. 2).
Another subset of NLRs trigger activation of the cysteine protease caspase 1 (REF. 35),
which leads to caspase 1-dependent pyroptosis and processing and release of the
inflammatory cytokines IL-18 and IL-13 (FIG. 2). TLRs and caspase 1-activating NLRs
often act in concert with TLR stimulation, resulting in enhanced susceptibility to NLR-
mediated caspase 1 activation in response to ATP treatment36 38 and Yersinia infection12.
TLRs and NOD1 and NOD2 also stimulate the production and intracellular accumulation of
pro-IL-133,34. Thus, TLRs and NOD1 and NOD2 prime cells to undergo caspase 1
activation and produce maximal IL-1 in response to subsequent cytosolic recognition of
host- or pathogen-derived danger signals.
Caspase-1-activating NLRs
NLR recognition of bacterial, viral and host molecules, as well as toxic foreign products, can
lead to the activation of caspase 1. The NLR protein NLRP3 (NACHT, LRR and PYD
domains-containing protein 3; also known as NALP3) responds to multiple stimuli,
including pore-forming toxins3840, extracellular ATP in the presence of various pathogen-
associated molecules38,41,42, uric acid crystals43, virus-associated DNA44, RNA45,
asbestos46 and ultraviolet B irradiation47. The mechanism by which NLRP3 detects this
divergent group of signals is unknown. Cellular potassium efflux is a common response to
many of these stimuli, and preventing potassium efflux blocks caspase 1 activation4850.
However, potassium efflux alone does not seem to be sufficient to trigger activation of
caspase 1 (REFS 48,51), and preventing potassium efflux also blocks caspase 1 activation
that is mediated by another NLR, NLRP1b (also known as NALP1b)20,52,53. This indicates
that potassium efflux may not directly signal for NLRP3-dependent caspase 1 activation, but
rather creates an environment that is favourable for ligand detection and/or caspase 1
activation49,52,54. It is possible that host cells respond to all of these stimuli by generating
one or more secondary factors that bind NLRP3, and further experiments are needed to
determine how NLRP3 directly recognizes or participates in the response to such a broad
range of molecules.
The NLR protein NLRC4 (NLR family CARD domain-containing protein 4; also known as
IPAF) mediates the recognition of diverse bacterial pathogens, which during infection reside
extracellularly (for example, Pseudomonas) or intracellularly (for example, Salmonella,
Legionella, Listeria and Shigella), and share similar requirements for the activation of
caspase 1. These pathogens deliver virulence determinants into host cells through
translocation systems that form conduits between the bacteria and host cell cytosol. The
same conduits, key to the pathogenesis of infection, also betray the presence of pathogens by
introducing flagellin into the host cell, where its recognition is facilitated by NLRC4 (REFS
23,5559). During infection with cytosolic pathogens, such as Listeria, secreted flagellin has
direct access to the cytosol, and a translocation system is not required60. Expression of
flagellin in the macrophage cytosol stimulates NLRC4-dependent pyroptosis61, suggesting
that NLRC4 directly recognizes flagellin; however, such an interaction has not been
demonstrated. Interestingly, NLRC4-dependent caspase 1 activation has been reported
during infection with Pseudomonas and Shigella mutants that do not produce flagellin62,63.
These studies suggest that NLRC4, like NLRP3, can respond to additional bacterial
components that remain unidentified.
The NLR NLRP1b recognizes cytosolic delivery of B. anthracis lethal toxin, a

metalloprotease that can cleave host mitogen-activated protein kinases (MAPKs). NLRP1b-
mediated caspase 1 activation is not due to structural recognition of the toxin itself, as lethal
toxin that contains a point mutation in the catalytic site, but retains its native structure, fails
to activate caspase 1 (REFS 20,64). Proteolytic activity of lethal toxin is required for
caspase 1 activation, but MAPK cleavage alone is not sufficient, suggesting that as-yet-
unidentified lethal toxin substrates are involved20. Proteasome activity is also required for
caspase 1 activation in response to lethal toxin treatment20,30,53, suggesting that a lethal
toxin-mediated alteration in proteasome function allows caspase 1 activation30.
Several NLR proteins, in addition to those described above, have been implicated in caspase
1 activation35. The NLR neuronal apoptosis inhibitory protein 5 (NAIP5) is required for
caspase 1 activation during infection with Legionella, but does not seem to be necessary for
all bacteria that activate caspase 1 through NLRC4 (REF. 61), and the exact role of NAIP5
in pyroptosis is unknown. Francisella requires ASC (apoptosis-associated speck-like protein
containing a CARD), but not NLRC4 or NLRP3, to stimulate caspase 1 activation24,38,
which implicates another NLR in the recognition of this pathogen.
The inflammasome
NLRs recognize their cognate host- or microorganism-derived danger signals and trigger
formation of a multiprotein complex called the inflammasome, which contains caspase 1
(REFS 35,65). NLRs that have encountered their signal undergo nucleotide-dependent
oligomerization using their nucleotide-binding domain66. Some NLRs, including NLRP3,

bind to the adapter protein ASC, which contains a caspase activation and recruitment
domain (CARD) and interacts with caspase 1 (REF. 35) (FIG. 3a). Other NLRs, such as
NLRC4, contain a CARD and can directly interact with caspase 1 when overexpressed67
(FIG. 3a). The association of caspase 1 within this complex allows its processing and
activation35.
It has been proposed that a single NLR mediates caspase 1 activation in response to a given
stimulus, and these complexes can be observed in vitro65,66. However, other data suggest
that interactions between multiple NLRs might contribute to inflammasome formation. For
example, NAIP5 can affect the ability of Legionella to stimulate NLRC4-dependent
activation of caspase 1 (REF. 61). NAIP5 can bind NLRC4 and contains a pathogen-sensing
leucine-rich-repeat (LRR) domain57, but its exact role in inflammasome formation is
unknown. NAIP5 does not seem to play a part in all NLRC4-containing inflammasomes, as
NAIP5 is not required for caspase 1 activation by Salmonella61. Similarly, both NLRP3 and
NLRC4 have a role in caspase 1 activation in response to Listeria infection60, pore-forming
toxins39 and ultraviolet B irradiation47. These data suggest that multiple sensors are present
in the same complex and function cooperatively to activate caspase 1. In addition, microbial
infection could lead to cell damage and release of host danger signals, such as uric acid and
ATP, that stimulate the activation of caspase 1. However, the release of these host ligands
by dying cells has not been shown in vivo. Thus, host cells encounter a barrage of caspase 1-
activating ligands and are endowed with a diverse sensor array to trigger the common
downstream response of pyroptosis efficiently20.
Inflammasomes were observed microscopically during Salmonella infection and treatment

with B. anthracis lethal toxin, and active caspase 1 was found to be located within a single
inflammasome complex as well as diffusely distributed throughout the cytoplasm20 (FIG.
3b). The adapter protein ASC can self-associate and form similarly sized complexes in the
absence of an NLR54, but the extent to which the self-association of ASC contributes to the
formation of NLR-containing inflammasomes is unknown (FIG. 3b). However, the fact that
Salmonella-mediated activation of caspase 1 is reduced in ASC-deficient macrophages68
suggests that ASC facilitates caspase 1 activation even though it is not absolutely required
for the binding of NLRC4 to caspase 1 (FIG. 3a). These data are consistent with the
formation of a single, large inflammasome, or aggregation of multiple complexes that
contain one or more NLRs, rather than many smaller complexes within a cell. The
localization of a large percentage of active caspase 1 within a single complex could limit
access to some caspase 1 substrates and allow recruitment of others by a mechanism that is
analogous to recruitment of substrates to the proteasome. By this model, regulatory proteins
could recruit substrates, control access to the proteolytic regions of the complex and alter the
enzymatic function of the complex to regulate substrate cleavage69. Similarly, the catalytic
activity of caspase 9 is enhanced when it is bound to the apoptosome, a multiprotein
complex that is involved in caspase 9 activation70. Defining the components of native
inflammasomes will provide insight into how this complex functions in its regulation of
caspase 1 activity.
Inflammasome components can also interact with proteins that activate alternative cellular
processes or forms of cell death. Autophagy has been observed during infection of
macrophages with Legionella71,72 and Francisella73, which can also induce caspase 1 under
other in vitro conditions23,24. Failure to induce robust caspase 1 activation owing to
suboptimal ligand production by the pathogen or host mutations does not result in
pyroptosis, but instead may allow inflammasome components to interact with other cell
death machinery and stimulate alternative cell death pathways23,72. ASC- and caspase 1-
deficient macrophages fail to activate caspase 1 in response to multiple stimuli, but are not
always protected from cell lysis, suggesting that the absence of caspase 1-dependent
pyroptosis allows other cell death processes to predominate, including pyronecrosis and
autophagy62,63,74,124. Infection with Shigella or Salmonella triggers caspase 1 activation in
wild-type macrophages, but in the absence of caspase 1, infected macrophages display

features of autophagy63,75. The induction of autophagy by Shigella requires the NLR protein
NLRC4, implicating NLR proteins in stimulation of both pyroptosis and autophagy63.
Caspase 1-dependent processes

Several caspase 1-dependent processes do not directly contribute to cellular demise, but
accompany the cell death process and contribute to the inflammatory nature of pyroptosis. In
addition, some events that are caspase 1-dependent can occur in the absence of cell death.
Caspase 1 activation can result in a combination of the following processes, which are
dictated by cell type as well as the nature and magnitude of the stimulus received.
IL-1 and IL-18 processing and secretion

The inflammatory cytokines IL-1 and IL-18 undergo caspase 1-dependent activation and
secretion during pyroptosis. IL-1 is a potent endogenous pyrogen that stimulates fever,
leukocyte tissue migration and expression of diverse cytokines and chemokines76. IL-18
induces IFN production and is important for the activation of T cells, macrophages and
other cell types77. Both IL-1 and IL-18 play crucial parts in the pathogenesis of a range of
inflammatory and autoimmune diseases76,77. Although neither cytokine is required for the
process of cell death37,78, their production contributes to the inflammatory response elicited
by cells undergoing pyroptosis. IL-1 and IL-18 lack secretion signals and their mechanism
of release has not been definitively determined. Formation of caspase 1-dependent pores in
the plasma membrane is temporally correlated with cytokine release in macrophages7,
suggesting that cytokine secretion occurs through these pores (FIG. 1). Interestingly, lysis is
not required for the release of activated IL-1 and IL-18, because pharmacological inhibition
of lysis does not prevent caspase 1-dependent pore formation or cytokine secretion7. Thus,
cytokine secretion and cell lysis are both downstream consequences of caspase 1-dependent
pore formation (FIG. 1).
Additional mechanisms of IL-1 and IL-18 release have also been described that occur in
the absence of cell lysis. Monocytes package active caspase 1 and cytokine substrates into
lysosomes79,80, and secretion of processed cytokines occurs through lysosome fusion with
the cell surface80 (FIG. 1). Although this is an elegant mechanism for cytokine secretion in
the absence of pyroptosis, recent evidence suggests this may be limited to monocytes81.
Release of cytokine-containing vesicles has also been observed in a range of cell types,
including dendritic cells, microglial cells and macrophages, during caspase 1 activation in
response to treatment with ATP8285. Two mechanisms have been proposed for vesicle
release: fusion of multivesicular bodies with the cell surface82 and direct budding of
microvesicles from the plasma membrane8385 (FIG. 1). Vesicle release has so far only been
observed in response to ATP stimulation, and surface microvesicle shedding results in a
significant reduction in cell size owing to loss of the plasma membrane83,85. By contrast, in
Salmonella- and Burkholderia-infected macrophages, cells increase in size as processed
cytokines are released7,18, suggesting that alternative mechanisms also mediate secretion of
IL-1 and IL-18.
Additional inflammatory cytokines

Caspase 1 activation is also required for maximal production of inflammatory cytokines
other than IL-1 and IL-18. Active caspase 1 has been shown to bind to and facilitate
secretion of IL-1 by an unknown mechanism5,86. A modest but significant reduction in
TNF and IL-6 secretion by caspase 1-deficient macrophages in response to

lipopolysaccharide stimulation has also been reported14,15,87. This is due to caspase 1-
mediated cleavage of the TLR adapter protein TIRAP (Toll/interleukin-1 receptor domain-
containing adapter protein; also known as MAL). Caspase 1-processed TIRAP signals more
efficiently, resulting in enhanced TNF and IL-6 production and macrophage activation in
response to TLR2 and TLR4 ligands87. Therefore, in addition to regulating the production of
IL-1 and IL-18, caspase 1 activation can also have a role in fine-tuning cytokine responses
to microbial stimuli.
Inhibiting growth of intracellular bacteria

Caspase 1 activation helps to restrict the growth of intracellular pathogens. In macrophages
that fail to trigger robust caspase 1 activation in response to Legionella infection, the
bacteria replicate within an endoplasmic reticulum-derived compartment that resembles an
immature autophagosome71. Infection of macrophages that more readily activate caspase 1
results in the rapid caspase 1-dependent delivery of Legionella to lysosomes and degradation
of the bacteria23,88. Caspase 1 activity also enhances the killing of mycobacteria by
stimulating trafficking of the bacteria to lysosomal compartments89. However, caspase 1 is
not required for the degradation of all bacteria88. Legionella, mycobacteria and other
pathogens produce virulence factors that modulate the trafficking of intracellular
compartments, and further experiments are required to determine how caspase 1 allows
macrophages to overcome these bacterial factors and contributes to the control of pathogen
replication in vivo.
Cell repair and survival

Caspase 1 activation fails to trigger pyroptosis in all cell types, and somewhat surprisingly,
epithelial cells use caspase 1 activation to prevent cell death39. Caspase 1 activation
stimulates lipid production and membrane repair in response to the pore-forming toxins
aerolysin and -toxin, and indeed inhibition of caspase 1 activity actually enhances cell
lysis39. This suggests that under certain conditions activation of caspase 1 could represent a
cellular survival mechanism.
The function of caspase 1 is analogous to the activities of other apoptotic caspases (caspases
3 and 8) in modulating the fate of certain cell types90. Low levels of apoptotic caspase
activation prevent autophagic cell death, regulate the proliferation and differentiation of B
and T cells, and control the maturation of dendritic cells90. Higher levels of activation of the
same apoptotic caspases result in the non-inflammatory elimination of these cells90.
Similarly, the magnitude of caspase 1 activation modulates the response to microbial stimuli
and host factors that warrant an inflammatory response. Low levels of active caspase 1
stimulate cell survival responses, control intracellular bacterial growth and mediate
inflammatory cytokine production. When caspase 1 activation passes a critical threshold
level, cells undergo pyroptosis and release inflammatory intracellular contents.
We propose that the level of caspase 1 activation tailors the host response to inflammatory
stimuli. In addition, the fate of cells with active caspase 1 could be controlled independently
of active enzyme levels by the subcellular localization of caspase 1. Restriction of active
caspase 1 to lysosomes by monocytes79,80 could sequester certain substrates to one
compartment for cleavage and release, while keeping cellular substrates that mediate cell
death in another. In vivo, minimizing pyroptosis and intravascular lysis of circulating
monocytes would probably be crucial to avoid an unfocused and potentially lethal systemic
inflammatory response. The function of active caspase 1 could also be regulated by its
localization within the cytosol. The confinement of active caspase 1 to a single focus within
the cell cytosol has been observed20,54 (FIG. 3b), and this restricted localization could limit
the access of the active enzyme to certain cellular substrates, as previously discussed. The
molecular decision to undergo pyroptosis could be modulated by the presence of death
effector proteins within a given cell type. Cells are not uniformly susceptible to this process:
several stimuli that trigger pyroptosis in macrophages and dendritic cells fail to do so in
epithelial cells39,91.
Caspase 1 in host response and disease pathology

Pyroptosis protects against infection and induces pathological inflammation. Although
caspase 1 activity and pyroptosis can have a role as a protective host response to infectious
diseases, exuberant or inappropriate caspase 1 activation and pyroptosis can be detrimental
(FIG. 4). Mutations in NLR proteins can lead to inappropriate caspase 1 activation, which is
associated with hereditary autoinflammatory syndromes92. Furthermore, caspase 1 is
involved in the pathogenesis of several diseases, including myocardial infarction4, cerebral
ischaemia93, inflammatory bowel disease94, neurodegenerative diseases95 and endotoxic
shock14, each of which are characterized by inflammation and cell death. Caspase 1
deficiency, or pharmacological inhibition, provides protection against the inflammation, cell
death and organ dysfunction that are associated with these diseases, making caspase 1 an
attractive therapeutic target. The protection afforded by caspase 1 deficiency against sepsis
and renal failure is not mimicked by neutralization of the cytokine targets, IL-1 and IL-18
(REFS 9698), suggesting that caspase 1 has an additional role in disease apart from
cytokine processing.
Caspase 1 activation helps to clear pathogens, such as Salmonella99,100, Shigella101,

Legionella23,57, Francisella24, Anaplasma phagocytophilum102 and Listeria103, during
infection in vivo in response to innate immune recognition of microorganism-associated
patterns. This phenotype cannot be attributed solely to IL-18 and IL-1 production. Mice
that are deficient in caspase 1 are more susceptible to Francisella than mice that lack both
IL-1 and IL-18, indicating that cell death itself, or other caspase 1-dependent processes,
contributes to the control of infection104.
Caspase 1 activation also influences the development of adaptive immune responses. In

conjunction with IL-12, IL-18 plays a major part in stimulating the differentiation of T
helper 1 (TH1)-type CD4+ T cells and enhancing their IFN production5,77. Caspase 1-
deficient mice infected with Candida albicans, Listeria or A. phagocytophilum have an
impaired TH1 response compared with wild-type mice102,103,105. CD4+ T cells generated in
caspase 1-deficient mice during infection shift towards a TH2 phenotype102,103,105, resulting
in impaired resistance to secondary infection by pathogens for which TH1-type responses are
required for immunity105. The ability of caspase 1 activation to enhance the development of
adaptive immune responses is supported by the finding that the non-microbial activators of
caspase 1 can act as adjuvants. Uric acid released from necrotic cells enhances cross-
presentation and generation of CD8+ T cells that are specific for exogenous antigens106.
Aluminium-containing adjuvants also stimulate caspase 1 activation107 and lead to TH2
CD4+ T cells and robust humoral immune responses108. Mice that cannot activate caspase 1
in response to aluminium-containing adjuvants fail to recruit inflammatory cells109 and
cannot stimulate TH2 CD4+ T-cell responses109,110. However, the role of caspase 1 in the
regulation of antibody production remains controversial109112. The contributions of
pyroptosis to host resistance are therefore multifaceted. Early in infection, caspase 1-
mediated processes, including, but not limited to, IL-1 and IL-18 production, lead to
activation and recruitment of immune cells and innate control of infection. During persistent
infection, continued caspase 1-dependent inflammation promotes the development of
appropriate adaptive immune responses that lead to the resolution of infection (FIG. 4).
Microbial regulation of caspase 1 activation

Active caspase 1 allows the host to control various microbial infections, so it is not
surprising that pathogens have evolved mechanisms to limit the activation of caspase 1 in
response to infection. Innate recognition is often limited to microbial patterns that are
required for pathogen survival, such as peptidoglycan, lipopolysaccharide, and nucleic
acids33,34. Flagellin, which is recognized by NLRC4, is not required for the survival or
virulence of Salmonella or Legionella in vivo23,113. Legionella and Salmonella use
translocation systems to modulate host cell function, but must also avoid introducing
flagellin into the cytosol through these translocation systems and stimulating pyroptosis.
Both organisms downregulate flagellin production during intracellular growth114,115, which
could provide a strategy to avoid pyroptosis, thereby limiting inflammation and allowing
continued intracellular replication of the bacteria.
There are multiple examples of pathogens that induce an alternative form of cell death,
effectively eliminating cells that would otherwise undergo pyroptosis and stimulate
pathogen clearance. Apoptosis kills macrophages by a process that results in the production
of anti-inflammatory factors and maintains membrane integrity, thereby preventing release
of inflammatory intracellular contents2. The activation of apoptotic caspase 3 also results in
cleavage of the caspase 1 substrate IL-18 at an alternate site, rendering it non-
inflammatory5. Yersinia can trigger apoptosis in naive macrophages and dendritic cells,
which effectively prevents inflammatory pyroptosis12 (FIG. 5). Pseudomonas strains that
produce the type III secretion system-secreted protein ExoU induce caspase 1-independent
necrosis, resulting in lysis but preventing the cleavage and release of IL-1 and IL-18 (REF.
62). However, 80% of clinical isolates are ExoU negative62, and instead trigger
pyroptosis58,59,62 (FIG. 5). Pseudomonas strains that express ExoU are more virulent62,
supporting the hypothesis that neutralizing macrophages before they have the opportunity to
activate caspase 1 benefits the bacteria during infection.
Pathogens also produce factors that can directly inhibit the activation of caspase 1.
Poxviruses are DNA viruses that replicate in the cytoplasm and would therefore be readily
detected by NLRP3. The poxvirus protein M13L-PYD binds ASC through its pyrin domain
(FIG. 3a), thereby disrupting inflammasome formation and preventing binding to and
activation of caspase 1. Deletion of this viral protein results in increased caspase 1 activity
and impaired replication in host cells in vitro and during infection in vivo116. The influenza
virus protein NS1 has also been shown to limit caspase 1 activation and cell death by an
unknown mechanism117, which indicates that inhibition of caspase 1 activation could be a
common strategy for successful viral pathogens. Yersinia translocates type III secretion
proteins that counteract the caspase 1 activating potential of the type III secretion system
itself. Yersinia strains that lack all the type III secretion system-translocated proteins have an
increased ability to activate caspase 1 (REFS 12,91,118). Analysis of individual effectors
suggests that YopE has an important role in the inhibition of caspase 1 activation, probably
owing to the ability of YopE to modulate host Rho GTPase function118. Francisella
mutants that trigger induction of pyroptosis more quickly than the wild type have been
identified, suggesting that Francisella also possesses a mechanism for inhibiting caspase 1
(REF. 119), and Mycobacterium tuberculosis produces a zinc metalloprotease that prevents
activation of caspase 1 through an unknown mechanism89. Finally, mutants of Francisella
and Mycobacterium that cannot control caspase 1 activation are attenuated in vivo, which is
consistent with the idea that increased levels of active caspase 1 and pyroptosis limit
bacterial replication89,119 (FIG. 5).
Host cell activation redirects cell death

Caspase 1 activation clearly functions as a host defence mechanism in a wide range of
microbial infections. Although localized inflammation during infection could enhance tissue
disruption and pathogen dissemination, as infection progresses, caspase 1 activation limits
pathogen replication, enhances innate and adaptive immune responses, and improves host
survival (FIG. 4). Pathogens require mechanisms for preventing or controlling the potent
inflammatory outcome of pyroptosis to persist and cause disease. Likewise, the host has
evolved mechanisms to counteract pathogen-mediated regulation of caspase 1 activity and
successfully control infection. Activation of macrophages counteracts Yersinia-mediated
inhibition of pyroptosis12 and enhances susceptibility to Francisella-induced pyroptosis120.
Host recognition of microbial infection may lead to upregulation of NLRs or other unknown
accessory proteins that are involved in caspase 1 activation and prime macrophages to
undergo pyroptosis. This enhanced sensitivity to pyroptosis allows a shift from the non- or
anti-inflammatory modes of cell death triggered by Yersinia and Francisella in naive
macrophages (apoptosis and autophagy, respectively) to inflammatory pyroptosis (FIG. 5).
The transition from autophagy to pyroptosis is also observed during Legionella infection,
perhaps owing to increased production of flagellin by the bacteria72. The ability of
macrophage activation to enhance pyroptosis in response to Legionella infection remains
unexplored. Activation could sensitize Legionella-infected cells to undergo pyroptosis in
response to lower amounts of flagellin. Together, these data clearly indicate that a host-
mediated redirection of cell death to pyroptosis can benefit the host by increasing
inflammation and facilitating the resolution of infection.
Concluding remarks
A wide range of host and microbial factors stimulate caspase 1 activation, and this leads to
an array of caspase 1-dependent processes that include cell death, modulation of
inflammatory cytokine production and restriction of pathogen replication. Together, these
caspase 1-dependent processes benefit the host in vivo by contributing to the control of
microbial infection. Pathogens use virulence factors to limit caspase 1 activation, but the
host has mechanisms for priming cells to activate caspase 1 in the presence of this
inhibition. Ultimately, there is competition between host and pathogen to regulate caspase 1
activation, and the outcome dictates life or death of the host.
Host and microbial factors that trigger caspase 1 activation, and the host NLR proteins that
detect these molecules, have been the focus of recent research. We are only beginning to
understand the molecular mechanism of pyroptosis and other processes downstream of
caspase 1 activation. Identification of proteins cleaved by caspase 1 in vivo will probably
provide a great deal of insight and allow a more thorough mechanistic description of this
process. The localization or composition of the inflammasome could have some role in
regulating protein processing by caspase 1. It remains to be determined whether the
inflammasome complex can determine the fate of cells that have active caspase 1.
Importantly, the physiological features downstream of caspase 1 activation, including
pyroptosis, are conserved responses to multiple stimuli. Pyroptosis and other caspase 1-
dependent processes are therefore relevant to our understanding of important beneficial host
responses as well as medical conditions for which inflammation is central to the
pathophysiology of disease.
DATABASES
Entrez Genome Project
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprj
Anaplasma phagocytophilum | Bacillus anthracis | Candida albicans |

Mycobacterium tuberculosis
UniProtKB
http://www.uniprot.org
ASC | caspase 1 | caspase 3 | caspase 6 | caspase 8 | caspase 9 | IFN | IL-1 | IL-1 | IL-6
| IL-8 | IL-18 | NAIP5 | NLRC4 | NLRP1b | NLRP3 | NOD1 | NOD2 | TNF | TIRAP
FURTHER INFORMATION
Brad T. Cooksons homepage
http://depts.washington.edu/mcb/facultyinfo.php?id=190
ALL LINKS ARE ACTIVE IN THE ONLINE PDF
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
S.L.F. was supported by National Institutes of Health Grants AI47242 and P50 HG02360 and Poncin and
Achievement Rewards for College Scientist Fellowships. T.B. was supported by National Institute of General
Medical Sciences Public Health Service National Research Service Award Grant T32 GM07270 and a Helen
Riaboff Whitely Fellowship.
Abbreviations
Caspases A group of cysteine proteases that, based on their physiological roles,
can be divided into two groups: those involved in the initiation and
execution of apoptosis (caspase 2, 3, 6, 7, 8, 9 and 10) and those that
trigger inflammation (caspase 1 and related caspases).
Autophagy A programme of cellular self-digestion in which cytoplasmic
components are sequestered and degraded intracellularly in
autophagosomes. Autophagic cell corpses are ultimately removed by
phagocytosis.
Oncosis A caspase-independent pathway of cell death triggered by exposure to
toxins or physical damage that features organelle and cell swelling and
culminates in cell lysis with release of intracellular contents that
stimulate inflammatory responses.

Toll-like A transmembrane protein that contains a leucine-rich repeat domain and
receptor mediates host recognition of pathogen- and danger-associated
molecular patterns located in the extracellular milieu or within
endosomes.
Nod-like A protein that contains a leucine-rich repeat domain and mediates host
receptor recognition of pathogen- and danger-associated molecular patterns in
the host cell cytosol.
Proteasome A multiprotein complex that recognizes and degrades polyubiquitinated
substrates.
Pyronecrosis Results from Shigella infection at high MOI (multiplicity of infection),

morphologically resembles oncosis and is NLRP3-dependent and
caspase 1-independent.
Microvesicle A membrane vesicle of less than 0.5 m in diameter that is shed from
the plasma membrane of eukaryotic cells.
Necrosis Does not indicate a specific pathway of cell death, but is a post-mortem
description of dead cells that have reached equilibrium with their
surroundings.
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Figure 1. Pyroptosis, an inflammatory host response

Caspase 1 is cleaved and activated in response to multiple stimuli, but once activated,
caspase 1 results in a conserved programme of cell death referred to as pyroptosis. Caspase
1 activation also leads to rapid formation of plasma-membrane pores with a diameter of 1.1
2.4 nm. These pores dissipate cellular ionic gradients, allowing water influx, cell swelling
and osmotic lysis. The pro-forms of interleukin-1 (IL-1) and IL-18 are processed by
caspase 1 and released during pyroptosis, although the exact mechanism of secretion
remains controversial. Secretion does not require lysis and is temporally associated with
caspase 1-dependent pore formation, suggesting that these pores facilitate cytokine release.
Other suggested secretion mechanisms include caspase 1-independent lysosome exocytosis
and microvesicle shedding. Caspase 1 activity results in cleavage of chromosomal DNA by
an unidentified endonuclease. Cleavage of DNA does not result in the oligonucleosomal
fragments observed during apoptosis. Nuclear condensation is also observed but nuclear
integrity is maintained, unlike the nuclear fragmentation observed during apoptosis.
Figure 2. Sensing of host- and microorganism-derived danger signals leads to two distinct
outcomes: cellular activation and cell death
Leucine-rich repeat (LRR) domains mediate host recognition of pathogen- and danger-
associated molecular patterns. Toll-like receptors (TLRs) are LRR-containing
transmembrane proteins that detect danger signals located in the extracellular milieu and
within endosomes. TLRs initiate a signalling cascade that leads to cellular activation
(through nuclear factor-B (NF-B)-, mitogen-activated protein kinase (MAPK)- and

interferon (IFN)-regulatory factor (IRF)-dependent pathways) and inflammatory cytokine
production (including IFN, IFN, tumour necrosis factor (TNF), interleukin-12 (IL-12),
IL-6, IL-8 and pro-IL-1). Nod-like receptors (NLRs) function in the recognition of danger
signals introduced into the host cell cytosol. Like TLRs, NOD1 (nucleotide-binding
oligomerization domain-containing protein 1) and NOD2 stimulation results in cytokine
production. Another subset of NLRs mediate activation of the cysteine protease caspase 1,
which triggers caspase 1-dependent cell death (pyroptosis) and processing and release of the
inflammatory cytokines IL-18 and IL-1. NLRC4, NLR family CARD domain-containing
protein 4; NLRP3, NACHT, LRR and PYD domains-containing protein 3. NLRP1b,
NAHCT, LRR and PYD domains-containing protein 1b.
Figure 3. Components of the inflammasome and visualizing the inflammasome complex

a | Nod-like receptor (NLR) leucine-rich repeat (LRR) domains are implicated in sensing a
range of intracellular danger signals. After ligand recognition, the nucleotide-binding and
oligomerization domain (NBD) mediates nucleotide-dependent self-association of NLRs.
Some NLRs, such as NLRP3 (NACHT, LRR and PYD domains-containing protein 3; also
called NALP3), contain a pyrin (PYD) domain that interacts with the adapter protein ASC
(apoptosis-associated speck-like protein containing a CARD). ASC contains a caspase
activation and recruitment domain (CARD) that binds and facilitates activation of caspase 1.
Other NLRs, such as NLRC4 (NLR family CARD domain-containing protein 4; also known
as IPAF), contain a CARD and can directly interact with caspase 1. However, ASC is often
required for NLRC4-dependent caspase 1 activation, indicating that ASC may participate in
NLRC4 inflammasome formation or play an additional part in caspase 1 activation. b |
Salmonella (red) infection of macrophages results in activation of caspase 1 (green), which
is visualized here using a fluorescently labelled inhibitor of the active enzyme. Active
caspase 1 is often concentrated within a single focus (indicated by the arrow) and diffusely
distributed throughout the cytoplasm. A similar distribution of active caspase 1 is seen in
macrophages treated with Bacillus anthracis lethal toxin20.
Figure 4. Caspase 1 activation in health and disease: fighting infection versus pathological
inflammation
Caspase 1 plays a protective part in the response to microbial infection. a | In response to
infection, quiescent cells undergo caspase 1 activation and pyroptosis, allowing cleavage
and release of interleukin-18 (IL-18), IL-1 and other inflammatory intracellular contents.
Quiescent cells can also undergo activation in response to inflammatory mediators, thereby
lowering the threshold for caspase 1 activation and pyroptosis and stimulating increased
production of IL-1. b | As infection progresses, the inflammation that occurs as a
consequence of pyroptosis leads to an increased population of activated cells that are primed
to undergo pyroptosis and have increased inflammatory potential. c | Inflammatory contents
produced during pyroptosis recruit and activate immune cells and stimulate the development
of adaptive immune responses. This contributes to the control and ultimate resolution of
microbial infection, and returns tissues to their resting state. Alternatively, caspase 1
activation can be detrimental, as mutations in Nod-like receptor (NLR) proteins or the
persistence of sterile inflammatory stimuli can result in inappropriate and/or excessive
caspase 1 activation. The inflammation produced by this process increases the population of
activated cells that are primed to undergo pyroptosis and express increased levels of IL-1,
and the amplification cycle persists (b). This potentiates the response and maintains an
inflammatory state, which, if uninterrupted, leads to pathology.
Figure 5. Susceptibility to pyroptosis is governed by pathogen and host modulation of caspase 1

activation
Pathogens have mechanisms for modulating cell death by inhibiting caspase 1 activation or
inducing an alternative form of cell death that is more conducive to their continued
replication. Yersinia (a) and Pseudomonas (b) translocate type III secretion effectors,
resulting in apoptosis and necrosis, respectively. Pathogens can fail to induce robust caspase
1 activation owing to suboptimal danger signal production by the pathogen (c). In addition,
host mutations may not allow sufficient levels of caspase 1 activation to trigger pyroptosis.
These infected macrophages often display features that are consistent with autophagy.
Robust production of caspase 1-activating ligands by Legionella during infection of a
susceptible macrophage triggers pyroptosis (d). Not all cells are uniformly susceptible to
pyroptosis, and macrophage activation enhances caspase 1 activation (FIG. 4) in response to
Yersinia and Francisella infection, which do not stimulate pyroptosis in naive macrophages.

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Nat Rev Microbiol. 2009 February ; 7(2): 99109. doi:10.1038/nrmicro2070.

Pyroptosis: host cell death and inflammation

Tessa Bergsbaken*, Susan L. Fink, and Brad T. Cookson*,

Department of Molecular and Cellular Biology, University of Washington, Seattle, Washington

Correspondence to B.T.C. cookson@u.washington.edu.

Mechanism and features of pyroptosis

Pyroptosis features rapid plasma-membrane rupture and release of proinflammatory

Bacillus anthracis produces plasma-membrane pores with a functional diameter of 1.12.4

Box 1 | Apoptosis is a programmed process that results in non-inflammatory cell

TLRs and NLRs

The NLR NLRP1b recognizes cytosolic delivery of B. anthracis lethal toxin, a

oligomerization using their nucleotide-binding domain66. Some NLRs, including NLRP3,

Inflammasomes were observed microscopically during Salmonella infection and treatment

wild-type macrophages, but in the absence of caspase 1, infected macrophages display

Caspase 1-dependent processes

IL-1 and IL-18 processing and secretion

Additional inflammatory cytokines

TNF and IL-6 secretion by caspase 1-deficient macrophages in response to

Inhibiting growth of intracellular bacteria

Cell repair and survival

Caspase 1 in host response and disease pathology

Caspase 1 activation helps to clear pathogens, such as Salmonella99,100, Shigella101,

Caspase 1 activation also influences the development of adaptive immune responses. In

Microbial regulation of caspase 1 activation

Host cell activation redirects cell death

Anaplasma phagocytophilum | Bacillus anthracis | Candida albicans |

stimulate inflammatory responses.

Pyronecrosis Results from Shigella infection at high MOI (multiplicity of infection),

from apoptosis to caspase-1-dependent pyroptosis. PLoS Pathog 2007;3:e161. [PubMed:

17. Kelk P, Johansson A, Claesson R, Hanstrom L, Kalfas S. Caspase 1 involvement in human

is dependent on the ASC/caspase-1 axis. J. Exp. Med 2005;202:10431049. [PubMed: 16230474]

cryopyrin/Nalp3. Nature 2006;440:233236. [PubMed: 16407888]

inflammasome components CIAS1/cryopyrin/NLRP3 and ASC. Cell Host Microbe 2007;2:147

81. Brough D, Rothwell NJ. Caspase-1-dependent processing of pro-interleukin-1 is cytosolic and

Microbe 2008;3:224232. [PubMed: 18407066]

Figure 1. Pyroptosis, an inflammatory host response

(through nuclear factor-B (NF-B)-, mitogen-activated protein kinase (MAPK)- and

Figure 3. Components of the inflammasome and visualizing the inflammasome complex

Figure 5. Susceptibility to pyroptosis is governed by pathogen and host modulation of caspase 1

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